1. Field of the Invention
This invention relates to a device for use in the liquid phase quantitative analysis of oligonucleotides, such as deoxyribonucleic acid (DNA) and the method of forming the device. The invention also relates to a DNA assay kit that includes the device and to the method of obtaining quantitative analyses by the use thereof. More particularly, the analysis, in its preferred form, entails using colorimetric techniques.
2. Information Disclosure Statement
In the past, the technology for liquid phase quantitative analysis of oligonucleotides, particularly DNA, has not been available. Most quantitative analysis of DNA has been in the solid phase either through column chromatography or filter media techniques such as the Downs and Wilfinger Method . [Ref. Anal. Blochem., 131, pp. 538-47 (1983).]
DNA molecules are double stranded helical molecules. Each strand consists of sequences of nucleic acids held together by phosphate linkages. The sequence of nucleotides determines the function of the molecule. The two strands are held together by base pair complementary binding, and each strand of DNA binds only to its complement. By using the proper conditions of pH and temperature, it is possible to split the double strand into two complementary single strands. The single stranded DNA can be used to identify the source of a particular material by comparing the sequence of nucleic acids with the sequence from known sources. Alternatively, the presence of a particular DNA can be detected by attempting to hybridize it to a complementary DNA of known sequence.
Most DNA analysis is carried out by hybridization studies on a membrane surface. In this technique, the DNA to be identified is immobilized onto the surface of a nitro cellulose membrane by spotting a drop of solution containing the single stranded DNA onto the membrane, sealing the membrane into a plastic bag and incubating overnight at elevated temperatures. Then a solution of the complementary DNA which has been tagged with radioactive I.sup.125 or p.sup.32 is placed onto the spot and incubated again to allow hybridization to take place. After incubation, the spot is washed carefully to remove any unbound material, and then the presence of radioactivity is measured. If the unknown strand and the radio labelled strand are complementary, radioactivity will be retained. The success of the procedure requires that the binding conditions and the hybridization condition be different enough to prevent any direct binding of the labelled DNA to the membrane. This procedure is at best a semiquantitative procedure, and in most cases is only qualitative because the area of the spot is not confined and well defined.
In preparing for this application, a search of the Chemical Abstracts was conducted. This search uncovered the following:
PCT Int'l. Appl.--WO 90 07582, Jul. 12, 1990, U.S. Appl. Ser. No. --293,895; PCT Int'l. Appl.--WO 88 02785, U.S. Appl. Ser. No.--919,201; and, an article by J. Lundeberg et al, entitled "Rapid Colorimetric Detection of In Vitro Amplified DNA Sequences" from DNA And Cell Biology, Vol. 9, No. 4, 1990. These references are of interest only.
In further preparation, a pre-examination patentability search was performed, which search reviewed several subclasses of Class 935, especially Subclasses 78 and 86. The search uncovered the following patents:
U.S. Pat. No. 4,767,699--C.P.H. Vary et al. (Allied Corp.) PA1 U.S. Pat. No. 4,889,798--E. Rabbani (Enzo Blochem Inc.) This patent describes a heterologous detection system for a biotinylated probe. The probe can be fixed to nitrocellulose paper, nylon, etc. PA1 U.S. Pat. Nos. 4,751,177 4,797,355--Y. Stabinsky (Amgen Inc.) PA1 U.S. Pat. No. 4.994,373--J. G. Stavrianopoulos et al. (Enzo Blochem, Inc.) PA1 U.S. Pat. No. 4,886,741--D. E. Schwartz (Microprobe Corp.) PA1 U.S. Pat. No. 4,888,274--C. M. Radding et al. (Yale University) PA1 U.S. Pat. No. 4,925,785--C.-N. J. Wang et al. (Biotechnica Diag. Inc.) Hybridization methods using a solid support bound to a nucleic acid sequence which can bond to a probe. In a heterogenous sandwich assay, a polymer-bound probe is restricted to a very small area on the support, to increase signal intensity from the label. PA1 U.S. Pat. No. 4,908,307--K. D. Rodland et al. PA1 EPA 0 390 500--David Okrongly (Applied Immunesciences, Inc.) PA1 WO A 8 607 387--David L. Snitman et al. (Amgen) PA1 EPA 0 192 168--Nanibrushan Dattagupta (Molecular Diagnostics, Inc.)
The probe described contains biotin, and the hybridized complex may be separated therefrom using a streptavidin-bound matrix. The probe therefrom is bound covalently or otherwise to a solid support: this may include binding with a biotin-avidin or streptavidin bridge.
A polynucleotide is synthesized on a solid support such as glass, and is then used for binding polynucleotide probes. (The glass described is amine-functionalized with a silane derivative).
Polynucleotide sequences to a glass support, which may have wells, e.g. by using ganuna-aminoproply-triethoxysilane. Biotin-avidin may be used to bond a signalling moiety to the probe.
Enzymatic detection of hybridized probes using biotin with an enzyme conjugated with avidin or streptavidin is discussed.
Biotinylated probes and target purification by streptavidin chromatography are described in examples.
Variants of conventional hybridization of DNA bound to nitrocellulose or nylon membranes are disclosed. Labeling by biotinylated adenosine polymer and detection using streptavidin and biotinylated enzyme are mentioned.
These patents are a typical reflection of the state-of-the-art of genetic engineering assay procedures, but do not generally reflect the application of the same to liquid phase systems or to quantitative analysis of oligonucleotides. These patents do not provide a means for nor do they teach toward forming or using a masked receptor as described hereinbelow or for the method of use thereof.
Further, the typical reflection of the state-of-the-art of immunoassay procedure, specifically as to covalent binding, is attained by reference to U.S. Pat. No. 4,778,767. There it is noted that Shekarchi, et al., J. Clin. Microbiology 16(6), 1012-1018 (December, 1982) discloses an immunoassay procedure wherein an immunoreagent is immobilized on a small stick, i.e., "microstick", for easy manipulation of the reagent and the immunocomplex. While a number of materials were investigated for use in such microsticks, including stainless steel, nylon, polycarbonate, polystyrene and polytetrafluorolethylene (PTFE), it was found that the PTFE, cleaned by the conventional procedure of rinsing with 6N HCl, absorbed very little of the immunoreagent as compared with the other materials and could not be used as a base for the immunoreagent until it had been coated with polycarbonate or nitrocellulose. The previously referenced patent further cites German Offenlegungsschrift No. 32 00 822, published Jul. 21, 1983, which discloses a method for activating the surface of PTFE articles, in order to bond immunoreagents covalently, by contacting the PTFE surface with an ammoniacal solution of sodium, followed by treatment with carbodiimide. It indicates that the process was apparently attempted because it was found that adsorption of the immunoreagent on PTFE was unsatisfactory, and also that there is some question whether the procedure of this German application actually can immobilize a useful amount of immunoreagent on PTFE. As are described in more detail below, other procedures for surface treatment of polystyrene have been adopted resulting in the commercial availability of polystyrene covalent binding plates.
In addition to the Syvanen et al. reference cited in the U.S. Application, an International Search Report of the corresponding application has brought to the inventor's attention the following:
The publication describes the covalent attachment of macromolecules on substrate surfaces, particularly uncross-linked polystyrene. The surface is functionalized employing hydroxymethylamides and is then useful for complex formation between complementary binding pairs.
This publication describes a kit for the isolation and detection of a selected target nucleic acid sequence. A solution hybridization with two probes, each complementary to a different portion of the target. After hybridization of the target, a solid support is employed to immobilize the target-probe hybrid complex.
This publication describes assay method for detecting the presence of a particular polynucleotide sequence in a test sample. After solution hybridization, the target probe hybrid is bound to an immobilized form of the reaction partner. The detection probe is labeled as desired.
In the field of oligonucleotide assays, the need for liquid phase, quantitative analysis has been a long-standing problem which is addressed by this invention.